The present invention relates to the field of Pax-encoding vectors and more particularly to vectors comprising sequences that encode Pax7, Pax3, and/or biologically active variants or fragments thereof, and their use to induce differentiation of adult stem cells to produce myoblasts. The present invention also relates to cells transformed with a nucleotide sequence encoding Pax proteins.
Myoblasts are precursor cells of the mesoderm that are destined for myogenesis. The determined myoblasts are capable of recognising and spontaneously fusing with other myoblasts leading to the production of a differentiated myotube. The multinucleated myotube no longer divides or synthesises DNA but produces muscle proteins in large quantities. These include constituents of the contractile apparatus and specialised cell-surface components essential to neuromuscular transmission.
Eventually, the differentiated muscle cell exhibits characteristic striations and rhythmic contractions. A further step in this pathway is maturation; the contractile apparatus and muscle at different stages of development contain distinct isoforms of muscle proteins such as myosin and actin, encoded by different members of multigene families.
Myoblasts have the potential for being used in a variety of ways. For example, the myoblasts may serve as vehicles for cell therapy, where one or more genes may be introduced into the myoblasts to provide a protein of interest. In order to find wide utility in therapeutic applications, however, it will be necessary to develop methods for the sustained production by myoblasts carrying the gene of interest.
Myoblasts are thought to be capable of repairing damaged or injured myofibers (Mauro, A., J. Biophys. Biochem. Cytol., 9: 493-495 (1961); Bischoff, R., in Mvology, Engel, A. G. and Franzini-Armstrong, C., Eds., New York: McGraw Hill, pp. 97-119, 1994; and Grounds, M., Adv. Exp. Med. Biol., 280: 101-104 (1990)). Because myoblasts are thought to be capable of repairing damaged or injured myofibers, the technique of myoblast transfer (myoblast transplantation) has been proposed as a potential therapy or cure for muscular diseases, including Duchenne muscular dystropy (DMD).
Myoblast transfer involves injecting myoblast cells into the muscle of a mammal, particularly a human patient, requiring treatment. Although developed muscle fibres are not regenerative, the myoblasts are capable of a limited amount of proliferation, thus increasing the number of muscle cells at the location of myoblast infusion. Myoblasts so transferred into mature muscle tissue will proliferate and differentiate into mature muscle fibres. This process involves the fusion of mononucleated myogenic cells (myoblasts) to form a multinucleated syncytium (myofiber or myotube). Thus, it has been proposed that muscle tissue which has been compromised either by disease or trauma may be supplemented by the transfer of myoblasts into the compromised tissue.
Moreover, cell cultures are widely used as in vitro models for studying the events involved during in vivo cellular or tissue development. For example, muscle developmental events can be reproduced during the myogenic differentiation of stem cell cultures. Accordingly, permanent mammalian cell cultures, especially human myogenic cell cultures, would be of considerable value for providing useful tools for dissecting the molecular and biochemical cellular events, for identifying and testing new drugs for muscular diseases, such as dystrophies, for the study of myogenesis, etc.
The “paired-box” family of transcription factors is intimately involved in the control of embryonic development. Different members of the Pax family of transcription factors appear to regulate the development and differentiation of diverse cell lineages during embryogenesis (see Table 1) (Mansouri et al., 1999; Mansouri et al., 1994; Noll, 1993; Strachan and Read, 1994). Pax7 and the closely related Pax3 gene belong to a paralogous subgroup of Pax genes based on similar protein structures and partially overlapping expression patterns during mouse embryogenesis (Goulding et al., 1991; Jostes et al., 1990). Interestingly, Pax3 gene plays an essential role in regulating the developmental program of MyoD-dependent migratory myoblasts during embryogenesis (Maroto et al., 1997; Tajbakhsh et al., 1997).
Pax7 and Pax3 proteins bind identical sequence-specific DNA elements suggesting that they regulate similar sets of target genes (Schafer et al., 1994). Furthermore, increased expression and gain-of-function mutations in both Pax3 and Pax7 are associated with the development of alveolar rhabdomyosarcomas indicating that both molecules regulate similar activities in myogenic cells (Bennicelli et al., 1999). However, Pax7 but not Pax3 is expressed in adult human primary myoblasts (Schafer et al., 1994). Interestingly, differential expression of alternatively spliced Pax7 transcripts correlates with muscle regenerative efficiency in different strains of mice (Kay et al., 1998; Kay et al., 1997; Kay et al., 1995; Kay and Ziman, 1999).
Skeletal muscle regeneration has long been considered to be mediated solely by monopotential skeletal muscle stem cells known as satellite cells (Bischoff, 1994; Charge and Rudnicki, 2004). However, recent studies have identified novel populations of adult stem cells in skeletal muscle. For example, “side-population” (SP) cells isolated from muscle tissue participate in the regeneration of skeletal muscle and give rise to satellite cells (Asakura et al., 2002; Gussoni et al., 1999). In vitro, muscle SP cells readily form hematopoietic colonies, but do not spontaneously differentiate into muscle cells unless cocultured with satellite cell derived myoblasts (Asakura et al., 2002).
Various cell surface markers have been employed to purify adult stem cell populations from skeletal muscle, including c-kit, Scal, CD34, and CD45 (reviewed by Charge and Rudnicki, 2004). Almost all muscle-derived hematopoietic progenitor and blood reconstitution activity is derived from CD45+ cells (Asakura et al., 2002; McKinney-Freeman et al., 2002). Muscle-derived CD45+ cells purified from uninjured muscle are uniformly non-myogenic in vitro and do not form muscle in vivo (Asakura et al., 2002; McKinney-Freeman et al., 2002). However, coculture and in vivo injection experiments indicate that CD45+ SP as well as CD45− SP cells possess myogenic potential (Asakura et al., 2002; McKinney-Freeman et al., 2002).
There is a need in the art for novel cells that are capable of differentiating into muscle cells. Further, there is a need in the art for methods of promoting myogenic specification of stem cells. There is also a need in the art for novel uses and methods of treating a subject having a disease with stem cells that are capable of differentiating into muscle cells.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims. The sub-claims disclose further advantageous embodiments of the invention.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.